Heterozygous germline deficiencies in DNA mismatch repair (MMR) genes cause hereditary non-polyposis colorectal cancer (HNPCC), one of the most common inherited cancer syndromes, whereas homozygous deficiency causes severe hematological malignancy. Defects in MMR also are detected in other sporadic cancers, suggesting that loss of MMR is a common etiologic factor in human tumorigenesis. At the cellular level, MMR ensures the stability of the genome through the correction of errors made during DNA replication and the recognition of several types of DNA damage, leading to growth arrest and cell death. The precise mechanism of these different functions is unknown. Particularly poorly defined are the MMR-dependent responses to DNA damage. Cells lacking MMR demonstrate an elevated rate of spontaneous mutation and resistance to cell killing induced by DNA damaging agents. However, the extent to which the different phenotypes associated with MMR deficiency contribute to cancer risk is not known. To elucidate the mechanisms and importance of MMR functions in the prevention of cancer, we propose a functional characterization of the MMR gene MLH1. We will define the phenotypic consequences of specific mutations in MLH1 through the analysis of isogenic cell lines expressing MLH1 variants (Aim 1). We will elucidate the mechanism underlying genetic deficiencies identified in Aim 1 using biochemical assays of mismatch-directed excision, error correction, and DNA damage processing (Aim 2). Finally, we will identify and characterize novel mutations in MMR genes using a unique genetic selection for dominant-acting mutations that interfere with MMR-dependent responses to DNA damage (Aim 3). Our analyses will increase understanding of the mechanisms of MMR, the consequences of specific MMR gene mutations, and the importance of different MMR functions for the prevention of cancer. Such knowledge will facilitate more rationally designed intervention strategies for the treatment and/or prevention of cancer associated with MMR deficiency. [unreadable] [unreadable]